The instant invention relates to a novel process for preparing antiviral vaccines, particularly anti-foot and mouth disease vaccine.
As already known, the different processes for preparing vaccines against viral diseases are comprised by a sequence of steps which, although different in accordance with particular viruses and processes selected, may be classified as follows:
1. one or more steps for virus production; PA0 2. one or more steps for virus inactivation; and PA0 3. one or more steps for vaccine formulation.
The object of the present process invention has novel basic features in virus production and inactivation steps, and also proposes other novel steps and complementary stages through the whole process as preferred alternatives.
In accordance with the present invention, virus production is higher than that obtained through other known processes basically due to the fact that animals used are previously radiated with an ionizing radiation dose determined experimentally.
On the other hand, virus inactivation also is improved as compared to that obtained through techniques of known processes, due to the fact that ionizing radiations are also used, which were experimentally determined. The reason why better results are obtained is that radiation is carried out over a viral suspension at a very low temperature (-40.degree. C. or lower), thus avoiding virus degradation through the action of enzymes present in the viral culture medium.
Foot and mouth disease virus easily reproduces in mice, rats, hamsters and rabbits not more than 8 days old. Sensitivity to virus decreases as the age of the animals increases; thus, in adult animals, even high viral doses do not produce noticeable alterations.
However, it is important to use organs of adult animals for viral production in order to diminish production costs.
In accordance with the process of the instant invention virus production is carried out in an animal previously subjected to immunodepression through ionizing radiations, thus becoming sensitive to foot and mouth disease virus even when adult, and in tissues which are normally not sensitive to the virus.
In prior art anti-foot and mouth disease vaccines virus production was carried out by infecting bovine cattle, which are particularly sensitive to the virus, and then removing the viral material present in the tongue epithelium.
The material thus obtained, besides being scarce, was subjected to frequent contamination; therefore, vaccine mass production was practically unfeasible due to the cost and operating difficulties.
Further, survival culture of bovine tongue epithelium obtained from animals killed in slaughterhouses as support for viral reproduction was introduced (Frankel method). This method is still now one of the most widely used in the industry for the reproduction of foot and mouth disease virus for preparing vaccines.
Nevertheless, bacterial contamination of survival cultures is frequent, the collection of epithelia under proper conditions is difficult and frequent errors take place when obtaining antigens due to epithelia contamination or degradation. Therefore, costs per unit vaccine are high.
More recently, BHK (Baby Hamster Kidney, line 21, clon 13) cell culture was introduced; cell cultures in suspension are used for virus reproduction.
Such a process requires the use of sophisticated devices in order to maintain cell multiplication (Synthetic or semi-synthetic means with the addition of fetal sera). Due to their high cost, fetal sera have been replaced by adult bovine serum, but the use of the latter causes the risk of neutralizing antibodies which, in many cases, completely prevents virus reproduction. Further, cell adaptation to suspension culture is required, as well as virus adaptation to its multiplication in these cell cultures, with corresponding risks of antigen modification. Lastly, expensive equipment is required in order to guarantee culture bacterial sterility.
The process of the instant invention, in the steps corresponding to the obtaining virus, is improved over the processes of the prior art, basically due to the fact that it transforms the whole animal into a medium appropriate for virus reproduction, this fact being valid also for adult rodents and ruminants.
In fact, prior to virus inoculation, the animal is completely irradiated with ionizing radiation in a dosage that is between a minimum value for assuring immunological depression and a maximum value for assuring animal survival until its death caused by the action of the highly proliferated virus. Optimum dosages were found to be between 700 and 2700 rads (according to the species).
It was also found that a period of time of 24-36 hours between irradiation and virus inoculation was convenient in order to obtain the best results.
Another important improvement is the use of antibiotics during the viral proliferation period, in order to avoid bacterial reproduction promoted by the immunodepression caused by radiations.
In this way, an economical, technologically simple, high quality viral antigen source is obtained, which also bears a low contamination risk and low antigen modification rates.
Since known methods of virus collection may be applied to some tissues or cultures, the following collection step is preferred.
This step comprises removing skin and viscerae from the host animal. Soft parts are removed from the rest of the cadaver and are crushed with the addition of saline solution; the mixture obtained should be visually homogeneous.
Lastly, a liquid phase is separated by means of filtration or centrifugation, thus obtaining the required active viral suspension.
In relation to virus inactivation, the known methods are based on the chemical action of some substances, such as acethylethylenimine, betapropiolactone, glycidaldehyde, ethylethylenimine, bis-ethylenimine (BEI) or formaldehyde. In such a process, the viral suspension should be kept at room temperature or higher temperatures for 24-48 hours.
Under these conditions, physical and chemical agents act to degrade the virus antigenic proteins.
On the contrary, radiation with ionizing radiations, in the mentioned dosages, carried out at extremely low temperatures (typically -70.degree. C., and always below 0.degree. C.) does not cause the mentioned enzymic reactions.
Virus radiation with ionizing rays is already known in the art, but not for the purpose of the instant invention, but for forming part of the basic investigations.
Ernest Pollard, U.S., has applied radiation for obtaining inactivation of some virus properties (Inactivation Theory).
Another known reference of prior art is that disclosed by William Ginoza, "Inactivation of viruses by ionizating radiations and by heat", Methods in Virology, IV, pages 139-205 (1968).
Nevertheless, none of the above references are related to the use of radiations for obtaining industrial production of vaccine.
Ionizing radiation used for radiating animals as well as for inactivating the viruses may be of the electromagnetic radiation type, such as X-ray or Gamma-ray, or of the particle radiation type, such as alpha-particles, positions, electrons, protons, neutrons or deuterons.
Whichever its origin, foot and mouth disease antigen has a rather poor immunological ability. Therefore the antigen should be improved by other substances capable of exerting immunity coadjutant action, in relation to power as well as to duration.
In accordance with known processes, aqueous or oily carriers may be used. The former include aluminum hydroxide and the duration of the immunity obtained is not longer than four months. The second are emulsions, but in this case known vaccines have relatively large droplets, and in some cases their stability is low, thus causing rupture of the emulsion.
With the process of the present invention it is possible to obtain a minimum immunity term of six months, thus reducing the vaccine administration to bovines to twice a year.
Therefore, the instant invention proposes a formulation comprising producing a "water in oil" type emulsion, the discontinous internal phase of which is constituted by the inactivated viral suspension and its external dispersion phase comprises a mineral oil.
The mineral oil, acting as immunity adjuvant, is of the liquid paraffin type, which is a mixture of different proportions of paraffins and naphtenes, but lacking cyclic hydrocarbons due to their probable carcenogenic action.
Another suitable oil is polybutene, which is an oily hydrocarbon prepared through polymerization of olefins, mainly comprised by methylated isobutenes of constant formula.
Whichever the selected oil may be, when the antigen is to be concentrated or the emulsion viscosity is to be lowered, the viral suspension is previously adsorbed to the aluminum hydroxide with 2% aluminum oxide.
In both cases, and in order to obtain a low viscosity, stable, homogeneous emulsion of the type "water in oil", having small droplets (of a diameter not larger than 0.1 micron) and easy to be obtained at an industrial scale, three emulgents are employed: a lipophilic emulgent, sorbitan sesquioleate of hydrophilic-lipophilic balance (HLB) 3.7; a hydrophilic emulgent, polyoxiethylene sorbitan mono-oleate, HLB 15, in the case of an oil emulsion of the viral solution, or HLB 10 when the viral suspension is incorporated to aluminum hydroxide; and n-butanol as co-surfactant.
The object is to obtain any of the following formulations:
______________________________________ 1. Liquid paraffin 40.50% Sorbitan sesquioleate 6.50 Polyoxiethylene sorbitan 1.00 mono-oleate, HLB 15: n-butanol 2.00 Inactive viral suspension 50.00 2. Polybutene 40.50 Sorbitan sesquioleate 6.50 Polyoxiethylene sorbitan 1.00 mono-oleate, HLB 15 n-butanol 2.00 Inactive viral suspension 50.00 3. Liquid paraffin 53.60-43.60 Sorbitan sesquioleate 2.00 Polyoxiethylene sorbitan 2.40 mono-oleate, HLB 10 n-butanol 2.00 Aluminum hydroxide plus 40.00-50.00 inactive viral suspension 4. Polybutene 53.60-43.60 Sorbitan sesquioleate 2.00 Polyoxiethylene sorbitan 2.40 mono-oleate, HLB 10 n-butanol 2.00 Aluminum hydroxide plus 40.00-50.00 inactive viral suspension ______________________________________
The steps involved in the process are the following:
(a) Mineral oil and emulgents are mixed into a tank at 4.degree. C.;
(b) viral suspension, pure for formulations 1 or 2, or mixed with aluminum hydroxide in the case of formulations 3 or 4 (stirring for two hours, 1 part aluminum hydroxide and 1-5 parts of viral suspension, in accordance with the concentration desired) is introduced into another tank at 4.degree. C., settling is allowed for 24 hours, and supernatant needed for returning to the original volume of aluminum hydroxide is removed;
(c) contents of both tanks are poured into a closed and sterile loop homogenizator, operating under high pressure and continuous flow;
(d) the product is collected into a third tank.